1. Microbiology and Infectious Disease
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A nanocompartment system contributes to defense against oxidative stress in Mycobacterium tuberculosis

  1. Katie A Lien
  2. Kayla Dinshaw
  3. Robert J Nichols
  4. Caleb Cassidy-Amstutz
  5. Matthew Knight
  6. Rahul Singh
  7. Lindsay D Eltis
  8. David F Savage
  9. Sarah A Stanley  Is a corresponding author
  1. Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, United States
  2. Department of Molecular and Cell Biology, Division of Biochemistry, Biophysics and Structural Biology, University of California, Berkeley, United States
  3. Department of Plant and Microbial Biology, University of California, Berkeley, United States
  4. Department of Microbiology and Immunology, The University of British Columbia, Canada
  5. School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, United States
Research Article
Cite this article as: eLife 2021;10:e74358 doi: 10.7554/eLife.74358
4 figures, 1 table and 20 additional files

Figures

Figure 1 with 1 supplement
Mtb produces endogenous nanocompartments that package a peroxidase.

(A) Schematic of the nanocompartment operon in Mtb that encodes the encapsulin shell protein (Cfp29) and the dye-decoloring peroxidase cargo protein (DyP). (B) Transmission electron microscopy (TEM) of Cfp29 encapsulin proteins purified following heterologous expression of the Mtb nanocompartment operon in E. coli. (C) Size distribution of Cfp29 protomers purified from E. coli. (D) Peptide counts from mass spectrometry analysis of endogenous nanocompartments purified from Mtb. (E) TEM of endogenous nanocompartments purified from Mtb. Peroxidase activity of (F) unencapsulated and (G) encapsulated DyP (5 nM) using ABTS (480 nM) as a substrate in the presence of H2O2 (480 nM) at varying pH levels (4.0–6.0) as reported by a change in the absorbance at 420 nm. neg, no added enzyme.

Figure 1—figure supplement 1
Nanocompartment purification and complementation strategies.

(A) Coomassie-stained SDS-PAGE of fractions collected during purification of nanocompartments heterologously expressed in E. coli: (1) ultracentrifugation pellet post-CellLytic B solubilization, (2) size-exclusion chromatography input, (3) lane 1 diluted, (4) lane 2 diluted, and (5) encapsulin fraction from size exclusion. (B) Coomassie-stained SDS-PAGE of sucrose fractions collected during purification of nanocompartments from wild-type Mtb lysates: (1) fraction containing assembled encapsulin nanocompartment complexes, (5) ladder (6), lane 1 boiled in SDS for 30 min to dissociate encapsulin nanocompartment into monomers. (C) DyP samples with or without addition of hemin were analyzed by SDS-PAGE. Samples were loaded either in their unboiled native state (left half) or heat-denatured by boiling at 95°C for 15 min. Addition of hemin yields a tetrameric DyP at 144 kDa. (D) Western blot for Cfp29 (arrow) and Ag85A control (star) from wild-type Mtb (lane 1) and Δoperon mutant (lane 2) lysates. (E) Complementation strategy schematic for DyP::Tn mutants (top). DyP::Tn mutants were transformed with ATc-inducible complementation constructs encoding the unencapsulated cargo protein (DyP), the encapsulin shell protein (Cfp29), or the nanocompartment operon (Operon). Lysates from each strain were used for nanocompartment purification. Sucrose fractions containing high molecular weight Cfp29 protomers were identified in complemented strains expressing the encapsulin shell and the operon (middle) and were analyzed using TEM (bottom).

Nanocompartments protect Mtb from oxidative stress in acidic environments.

(A) OD600 measurements of wild-type and DyP::Tn Mtb grown in 7H9 medium following exposure to H2O2 for 96 hr. Values reported are normalized to the untreated controls. CFU enumeration of wild-type and Mtb nanocompartment mutants grown in (B) standard 7H9 medium (pH 6.5) and (C–F) acidified 7H9 medium (pH 4.5) following exposure to oxidative stress (2.5 mM H2O2) for 72 hr. (G) Fluorescence emissions of wild-type and Δoperon Mtb expressing mrx1-roGFP exposed to 5 mM H2O2 at pH 4.5 in 7H9 medium for 60 min. Data are reported as a ratio of fluorescence emissions following excitation at 490 nm and 390 nm. Figures are representative of at least two (E, F) or three (A–D, G) independent experiments. p-Values were determined using unpaired t-test. *p<0.05, **p<0.01.

Susceptibility of Mtb nanocompartment mutants to oxidative and acid stress is mediated by free fatty acids.

(A) Transposon sequencing (Tn-seq) data showing normalized sequence reads per gene for all putative Mtb peroxidases, catalases, and superoxide dismutases and (B) lipid and cholesterol metabolism Mtb mutants that were significantly attenuated following 72 hr exposure to 2.5 mM H2O2 at pH 4.5. (C) CFU enumeration of wild-type Mtb and Δoperon mutants following 24 hr exposure to 2.5 mM H2O2 at pH 4.5 in Sauton’s minimal medium and (D) 72 hr exposure to 2.5 mM H2O2 at pH 4.5 in 7H9 medium prepared using fatty acid (FA)-free bovine serum albumin (BSA) ± oleic acid (150 µM). (E) Intrabacterial pH measurements of wild-type and Δoperon Mtb expressing pUV15-pHGFP following 20 min exposure to 5 mM H2O2 at pH 6.5 or pH 4.5. 7H9 medium was prepared with standard BSA or FA-free BSA. Figures are representative of at least two (D) or three (A–C, E) independent experiments. p-Values were determined using an unpaired t-test. *p<0.05, **p<0.01.

Nanocompartment mutants are attenuated for survival in macrophages and are more susceptible to pyrazinamide (PZA) treatment.

CFU enumeration of wild-type Mtb and (A) Δoperon or (B) DyP::Tn mutants during infection of murine bone marrow-derived macrophages. Macrophages were infected with a bacterial MOI of 1, and CFUs were enumerated immediately following phagocytosis and at days 2 and 4. Error bars are SD from four replicate wells. (C) CFU enumeration of wild-type and Mtb Δoperon mutants following 72 hr exposure to PZA (24 μg/mL) and H2O2 (2.5 mM) in acidified 7H9 medium (pH 5.5). Comp = Δoperon + pOperon. (D) Infection of BALB/C mice with WT and Δoperon mutant with and without treatment with 150 mg/kg PZA. CFU at 35 days post infection in the lung is shown. (A) and (B) are representative of at least five independent experiments; (C) is representative of three experiments and (D) is representative of two experiments. p-Values were determined using an unpaired t-test. **p<0.01; ***p<0.001.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mycobacterium tuberculosis)DypGenBankGene ID: 885388, Rv0799c
Gene (My. tuberculosis)Cfp29GenBankGene ID: 885460, Rv0798c
Strain, strain background (Mus musculus)BALB/CThe Jackson LaboratoryStock no: 000651
Strain, strain background (M. tuberculosis)H37RvEric Rubin Lab, Harvard School of Public Health
Strain, strain background (Escherichia coli)BL21 (DE3) LOBSTRkerafastCat# EC1002
Genetic reagent (M. tuberculosis)DyP::TnBroad Institute, Hung LabRv0799c::Tn
Genetic reagent (M. tuberculosis)ΔoperonThis studyΔRv0799c-Rv0798cSee Materials and methods
Genetic reagent (M. tuberculosis)ΔCfp29This studyΔRv0798cSee Materials and methods
Genetic reagent (M. tuberculosis)ΔDyPThis studyΔRv0799cSee Materials and methods
Recombinant DNA reagentPet14bNovagenCat# 69660-3
Recombinant DNA reagentpUV15tetORmAddgeneCat# 17975AHT-inducible
construct for all
complementation
Recombinant DNA reagentpKL4This studyRv0798c
cloned into
pUV15tetORm
(with KanR)
Recombinant DNA reagentpKL5This studyRv0799c
cloned into
pUV15tetORm
(with KanR)
Recombinant DNA reagentpKL6This studyOperon
cloned into
pUV15tetORm
(with KanR)
Recombinant DNA reagentpKL14This studyOperon
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpKL15This studyRv0798c
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpKL16This studyRv0799c
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpUV15 pHGFP HygR:AddgeneCat# 70045Rv0799c
cloned into
pUV15tetORm
(with HygR)
Recombinant DNA reagentpMV762-mrx1-roGFP2Amit Singh, ICGEB, IndiaPMC3907381
AntibodyAnti-Mtb Cfp29Rabbit polyclonalProduced by
GenScript USA,
see Materials
and methods
1:10,000
AntibodyHRPGoat anti-rabbit polyclonalSanta Cruz
Biotechnology
sc-2030
1:5000
Chemical compound, drug3-Ethylbenzothia
zoline-6-sulfonic acid
Millipore SigmaCat# 10102946001

Additional files

Transparent reporting form
https://cdn.elifesciences.org/articles/74358/elife-74358-transrepform1-v2.docx
Supplementary file 1

Mtb transposon sequencing screen in acidified broth with perxoide stress.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp1-v2.xlsx
Source data 1

Original scanned image of the gel depicting Figure 1—figure supplement 1A.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp2-v2.zip
Source data 2

Scanned image of the gel depicting Figure 1—figure supplement 1A.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp3-v2.zip
Source data 3

Original scanned image of the gel depicting Figure 1—figure supplement 1B.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp4-v2.zip
Source data 4

Original scanned image of the gel depicting Figure 1—figure supplement 1B.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp5-v2.zip
Source data 5

Original scanned image of the gel depicting Figure 1—figure supplement 1C.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp6-v2.zip
Source data 6

Original image capture of PVDF membrane to show the molecular weight ladder (Figure 1—figure supplement 1D).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp7-v2.zip
Source data 7

Original fluorescence image of western blot with Ag85B antibody (Figure 1—figure supplement 1D).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp8-v2.zip
Source data 8

Original fluorescence image of western blot with Cfp29 antibody (Figure 1—figure supplement 1D).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp9-v2.zip
Source data 9

Western images from Figure 1—figure supplement 1D.

https://cdn.elifesciences.org/articles/74358/elife-74358-supp10-v2.zip
Source data 10

Original scanned image of the gel depicting both Tn::DypB+DypB and Tn::DypB+Cfp29 (Figure 1—figure supplement 1E).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp11-v2.zip
Source data 11

Original scanned image of the gel depicting Tn::DypB+Operon (Figure 1—figure supplement 1E).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp12-v2.zip
Source data 12

This image depicts Tn::DypB+DypB, Tn::DypB+Cfp29, and Tn::DypB+Operon with molecular weight markers, lane labels, and the section of the image that is depicted in the main figure outlined in blue (Figure 1—figure supplement 1E).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp13-v2.zip
Source data 13

Wig file for control (library only) replicate 1 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp14-v2.zip
Source data 14

Wig file for control (library only) replicate 2 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp15-v2.zip
Source data 15

Wig file for control (library only) replicate 3 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp16-v2.zip
Source data 16

Wig file for experimental (H2O2_low pH) replicate 1 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp17-v2.zip
Source data 17

Wig file for experimental (H2O2_low pH) replicate 2 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp18-v2.zip
Source data 18

Wig file for experimental (H2O2_low pH) replicate 3 (Figure 3A and B).

https://cdn.elifesciences.org/articles/74358/elife-74358-supp19-v2.zip

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